A3.14 PD-1 Signalling and Low RORC mRNA Content Influence the TREG/TH17 Balance in Human Cord Blood

Abstract

<h3>Background and Objectives</h3> In neonates, the immune system is less responsive than in adults. Explanations for this involve differences in T helper (Th) 1 versus Th2 cell numbers and altered cytokine profiles after stimulation. We hypothesised that neonatal immune deviation may also involve FOXP3⁺ regulatory T cells (Treg) and Th17 cells. <h3>Materials and Methods</h3> In order to investigate this, we compared CD4⁺CD25^–CD45RO^– naïve T cells from human cord blood (CB) with cells from adult peripheral blood (APB). Cells were activated in vitro by anti-CD3 in the presence of viable antigen presenting cells (APB). The induction of regulatory T cells and Th17 cells was analysed by flow cytometry. Cytokine production was measured by multiplex immunoassays and RORC mRNA content by PCR. Specific interactions between APC and T cells were blocked with monoclonal antibodies during cell culture. <h3>Results</h3> CB cells show lower numbers of Treg ex vivo than APB. However, upon activation, high percentages of functionally suppressive FOXP3⁺ Treg are induced in CB. Replacement of viable APC by irradiated APC or anti-CD28 abrogated this effect, suggesting that live APC from CB in particular may imprint a Treg phenotype. Indeed, CB APC were able to induce high numbers of FOXP3⁺ cells in alloreactive immune reactions with APB T cells as well. Addition of blocking antibodies against CD80, CD86 or CTLA-4 to CB cultures did not affect the number of Tregs induced. Blockade of the PD-1/PD-Ligand (PD-L)1 interaction however showed a marked decrease in FOXP3⁺ cell numbers. CB cells showed a consistently reduced protein kinase B (PKB)/c-Akt phosphorylation upon activation. On top of that, in contrast to APB, low concentrations of APC derived pro-inflammatory cytokines were detected in CB cultures and no Th17 cells were induced. Addition of Th17 inducing cytokines reduced FOXP3⁺ cell numbers in CB, but did not induce IL-17 production. PCR of the Th17 defining transcription factor RORC showed a significantly reduced concentration of this protein in CB. <h3>Conclusions</h3> Human cord blood cells have an increased propensity to become FOXP3⁺ Treg as compared to APB. This effect is due to low production of inflammatory cytokines and PD-1/PD-L1 interaction. Inability to induce Th17 cells in CB could not be abrogated by Th17 inducing cytokines, but was due to reduced RORC mRNA content. This study shows mechanisms involving the human Th17/Treg induction switch that can also be important in inflammation and auto-immune disorders.